Erysiphe Necator Resistance Providing Genes in Vitis Vinifera

ABSTRACT

Provided herein are  Erysiphe necator  resistance conferring genes, plants, plant parts and seeds comprising the present resistance providing genes and the use thereof for selecting  Erysiphe necator  resistant plants. Specifically, provided herein are  Erysiphe necator  resistance conferring genes, wherein the amino acid sequence encoded by said resistance conferring genes is the primary amino acid sequence represented SEQ ID No. 1, or a primary amino acid sequence with more than 70% identity, preferably more than 80% identity, more preferably more than 90% identity, and most preferably more than 95% identity with SEQ ID No. 1; and wherein said resistance conferring gene is impaired.

The present invention relates to Erysiphe necator resistance conferringgenes, plants, plant parts and seeds comprising the present resistanceproviding genes and the use thereof for selecting Erysiphe necatorresistant plants.

Erysiphe necator, also designated as Uncinula necator, is a funguscausing powdery mildew disease symptoms in grape. The fungus is a commonpathogen for Vitis species of which the most important species is Vitisvinifera or grapevine.

Grapevine requires a huge amount of pesticides, particularly fungicides,to prevent yield losses. Between 1992 and 2003, 73% of the fungicidessold in France, Italy, Spain and Germany, were used for grapevineprotection, a crop that covers only 8% of the land used for agriculturein the considered countries (EUROSTAT, 2007).

Grapevine powdery mildew (PM) caused by the fungus Erysiphe necator, isone of the most economically relevant diseases of grapevine worldwide.E. necator is an obligate biotroph that can infect all green tissues ofgrapevine and causes significant losses in yield and berry quality. PMsymptoms are a white or grey powder covering of the upper and lowersurfaces of the leaves. Fruit infections result in shriveling orcracking of the berries. The quality of the fruit is severely damaged,with increased acidity and decreased anthocyanin and sugar content.

Powdery mildew is controlled with frequent applications of chemicalfungicides. However, the intense application of chemical fungicides hasseveral drawbacks. First of all, the effects on environment offungicides are well documented. Secondly, the costs of the chemicals andtheir applications can reach up to 20% of the total expenses for grapeproduction in some areas. Thirdly, the development of resistantpopulations of the pathogen was already documented by Baudoin et al.(2008) and Dufour et al. (2011), strongly reducing the efficacy ofchemical treatments. Therefore, there is increasing interest in thedevelopment of new alternative methods to chemical treatments.

The generation of PM-resistant varieties is one of the best options tomake sustainable grapevine cultivation a realistic possibility,preserving at the same time the incomes of the growers. A study carriedout on “Chardonnay” production in California, showed that the use ofPM-resistant variety could save to the growers around 720 $/ha, with asignificant reduction of fungicide usage (Fuller et al., 2014).

Most cultivars of the European grapevine (Vitis vinifera), whichincludes the world's finest and most widely planted wine and tablegrapevine cultivars, are highly susceptible to PM (Gadoury et al. 2003).In contrast, North American Vitis species co-evolved with E. necator andpossess various level of resistance to the pathogen (Fung et al., 2008).This resistance could be introgressed by crossing V. vinifera with oneof the resistant American Vitis species, but breeding is a slow processin grapevine and the acceptance of resistant hybrids by producers andconsumers has been limited in the past (Fuller et al., 2014). The use oftechnologies like genetic transformation or high-throughputmarker-assisted selection can be used to obtain resistant grapevinecultivars with desirable grape properties for producers and consumers(Feechan et al., 2013a).

The most common strategy to develop resistant plants is focused on theintrogression of resistance genes (R-genes). R-genes encode proteinsthat recognize pathogen effectors and trigger defense response, mediatedby a signaling network in which plant hormones play a major role (Pavanet al., 2010). Resistance is manifested as localized hypersensitiveresponse at the site of infection (Bari and Jones, 2009). Resistanceconferred by R-genes is scarcely durable, as mutations of pathogeneffectors, allow it to overcome resistance (Parlevliet et al., 1993).

An alternative approach is based on the inactivation of susceptibilitygenes (S-genes), defined as genes whose loss-of-function results inrecessively inherited resistance (Pavan et al., 2010). Some pathogensare able to suppress plant defense by activating plant proteins whichfunction is the negative regulation of plant immunity system. The genesencoding these plant proteins are known as susceptibility genes(S-genes) and their knock-out release the suppression of plant defenseand lead to resistance (Pavan et al., 2010). The disadvantage of S-genesis the pleiotropic phenotypes sometimes associated to their knock-out(Pavan et al. 2011). Mildew Locus O (MLO) genes are a typical example ofPM S-genes.

Resistance due to the knock-out of an MLO gene (mlo resistance) wasdiscovered in barley in 1992 (Jørgensen, 1992) and for a long time wasconsidered as a unique form of resistance. However, further studiesrevealed that MLO genes are largely conserved across plant kingdom andtheir loss-of-function resulted in resistance in several species, suchas Arabidopsis (Consonni et al., 2006), pea (Pavan et al., 2011), tomato(Bai et al., 2008) and pepper (Zheng et al., 2013). Not all MLO genesare S-genes and MLO family members are divided in seven clades(Acevedo-Garcia et al., 2014; Pessina et al., 2014). Only two cladescontain S-genes: clade IV contains all monocots S-genes (Panstruga etal., 2005; Reinstädler et al., 2010); and clade V contains all dicotsS-genes (Consonni et al., 2006; Bai et al., 2008; Feechan et al., 2008;Winterhagen et al., 2008). Not all the members of clades IV and V areS-genes.

Considering the economic impact of an Erysiphe necator infection ongrape production, there is a continuing need in the art for Erysiphenecator resistance providing genes.

It is an object of the present invention, amongst other objects, to meetthis need of the art.

According to the present invention, the above object, amongst otherobjects is met by providing impaired Erysiphe necator resistanceproviding genes as outlined in the appended claims.

Specifically, the above object, amongst other objects, is met accordingto a first aspect of the present invention by providing Erysiphe necatorresistance conferring genes, wherein the amino acid sequence encoded bythe resistance conferring gene is the primary amino acid sequencerepresented by SEQ ID No. 1, or a primary amino acid sequence with morethan 70% identity, preferably more than 80% identity, more preferablymore than 90% identity, and most preferably more than 95% identity withSEQ ID No. 1 under the condition that the present resistance conferringgenes are impaired.

In the alternative, the above object, amongst other objects, is metaccording to a first aspect of the present invention by providingErysiphe necator resistance conferring genes, wherein the amino acidsequence encoded by the resistance conferring gene is the primary aminoacid sequence represented by SEQ ID No. 2, or a primary amino acidsequence with more than 70% identity, preferably more than 80% identity,more preferably more than 90% identity, and most preferably more than95% identity with SEQ ID No. 2 under the condition that the presentresistance conferring genes are impaired.

As yet another alternative, the above object, amongst other objects, ismet according to a first aspect of the present invention by providingErysiphe necator resistance conferring genes, wherein the amino acidsequence encoded by the resistance conferring gene is the primary aminoacid sequence represented by SEQ ID No. 3, or a primary amino acidsequence with more than 70% identity, preferably more than 80% identity,more preferably more than 90% identity, and most preferably more than95% identity with SEQ ID No. 3 under the condition that the presentresistance conferring genes are impaired.

Sequence identity as used herein is defined as the number of identicalconsecutive aligned nucleotides, or amino acids, over the full length ofthe present sequences divided by the number of nucleotides, or aminoacids, of the full length of the present sequences and multiplied by100%. For example, a sequence with 80% identity to SEQ ID No. 1comprises over the total length of 539 amino acids of SEQ ID No. 1, 431or 432 identical aligned amino acids, i.e., 430 or 431/539*100%=80%.

An impaired resistance conferring gene according to the presentinvention is meant to indicate a gene providing a reduced, or evenabsent, susceptibility to Erysiphe necator as indicated by powder-likespots on the leaves and stems.

Impaired resistance conferring genes according to the present inventionare mutated genes. The mutation, or mutations, in the present genes canresults/result in impairment by different mechanisms. For example, oneor more mutations in protein encoding DNA sequences can result inmutated, truncated or non-functional proteins. One or more mutations innon-coding DNA sequences can cause alternative splicing, translation orprotein trafficking. Alternatively, one or more mutations resulting inan altered transcriptional activity of a gene, which determines theamount of mRNA available for translation to protein, can result in aresistance due to a low level, or complete absence, of encoded proteins.Additionally, the impairment of the present genes may be caused aftertranslation, i.e. at protein level.

Impaired is also indicated herein as encoding a non-functional gene orprotein. Although the function of the present genes is not yetidentified, a non-functional gene or protein can be readily determinedby establishing Erysiphe necator resistance (non-functional) or Erysiphenecator susceptibility (functional) in a plant. An Erysiphe necatorresistance (non-functional) plant is indicated by comprising a genewhich is mutated at the protein level as compared to the SEQ ID Nos. 1or 2 or 3 or reduced levels are observed of mRNA comprising SEQ ID Nos.4 or 5 or 6.

Functional and non-functional genes, or proteins, can also be determinedusing complementation experiments. For example, transforming an Erysiphenecator resistant Vitis vinifera plant with a copy the present genesunder the control a constitutive promoter will result in an Erysiphenecator susceptible Vitis vinifera plant.

According to the present invention, the present Erysiphe necatorresistance conferring genes provide Erysiphe necator resistance whenimpaired. Impaired according to the present invention can be indicatedby the absence, or decrease of a protein identified herein by SEQ IDNos. 1 or 2 or 3. In the art, many mechanisms are known resulting in theimpairment of a gene either at the transcription, translation or proteinlevel.

For example, impairment at the transcription level can be the result ofone or more mutations in transcription regulation sequences, such aspromoters, enhancers, initiation, termination or intron splicingsequences. These sequences are generally located 5′ of, 3′ of, or withinthe coding sequences represented by SEQ ID Nos. 4 and 5 and 6.Impairment can also be provided by a deletion of, rearrangement of orinsertion in the present genes.

Impairment at the translation level can be provided by a prematurestop-codons or other RNA to protein controlling mechanisms orpost-translational modifications influencing, for example, proteinfolding or cellular trafficking.

Impairment at the protein level can be provided by truncated, misfoldedor disturbed protein-protein interactions.

Independent of the underlying mechanism, impairment according to thepresent invention is indicated by a decrease, or absence, a functionalprotein according to SEQ ID Nos. 1 or 2 or 3.

Considering the above, according to an embodiment of the first aspect ofthe present invention, impairment according to the present inventioncomprises one or more mutations in the present genes resulting in theabsence of a protein expression product with a primary amino acidsequence represented by SEQ ID No. 1 or an mRNA comprising SEQ ID No. 4;or, in the alternative the absence of a protein expression product withthe primary amino acid sequence represented by SEQ ID No. 2 or 3 or anmRNA comprising SEQ ID No. 5 or 6, respectively.

According to another embodiment of this first aspect of the presentinvention, the present impairment comprises one or more mutations in thepresent genes resulting in a non-functional protein expression product.

According to still another embodiment of this first aspect of thepresent invention, the present impairment comprises a reducedtranscription level resulting in a reduced level of an mRNA comprisingSEQ ID No. 4 or SEQ ID No. 5 or SEQ ID No. 6.

According to yet another embodiment of this first aspect of the presentinvention, the present impairment comprises a reduced translation levelof an mRNA comprising SEQ ID No. 4 or SEQ ID No. 5 or SEQ ID No. 6.

According to an especially preferred embodiment of the invention, thepresent Erysiphe necator resistance conferring gene is derived fromVitis vinifera.

According to a second aspect, the present invention relates to Vitisvinifera plants comprising in their genome an impaired Erysiphe necatorresistance conferring gene as described above wherein the impairmentprovides Erysiphe necator resistance.

According to a preferred embodiment of this second aspect of the presentinvention, the present Vitis vinifera plants show an expression, ortranscription, of the present Erysiphe necator resistance conferringgenes being reduced by at least 10% as compared to a Vitis viniferaplant susceptible to Erysiphe necator, preferably wherein theexpression, or transcription is reduced by at least 20% as compared to aVitis vinifera plant susceptible to Erysiphe necator, preferably atleast 30%, more preferably at least 50%, even more preferably at least70%, and most preferably at least 80% such as 25%, 35%, 40%, 45%, 55%,60%, 65% or 75%.

According to another preferred embodiment of this second aspect of thepresent invention, the present Vitis vinifera plants display an absentexpression, or transcription of the present Erysiphe necator resistanceconferring genes.

According to an especially preferred embodiment of this second aspect ofthe present invention, the present Vitis vinifera plants comprise intheir genome an impaired Erysiphe necator resistance conferring geneencoding a protein with the primary amino acid sequence of SEQ ID No. 1,or a primary amino acid sequence with more than 70% identity, preferablymore than 80% identity, more preferably more than 90% identity, and mostpreferably more than 95% identity with SEQ ID No. 1; and, in addition,an impaired Erysiphe necator resistance conferring gene encoding aprotein with the primary amino acid sequence of SEQ ID No. 2, or aprimary amino acid sequence with more than 70% identity, preferably morethan 80% identity, more preferably more than 90% identity, and mostpreferably more than 95% identity with SEQ ID No. 2; and/or an impairedErysiphe necator resistance conferring gene encoding a protein with theprimary amino acid sequence of SEQ ID No. 3, or a primary amino acidsequence with more than 70% identity, preferably more than 80% identity,more preferably more than 90% identity, and most preferably more than95% identity with SEQ ID No. 3. Formulated differently, the presentinvention relates according to an especially preferred embodiment toVitis vinifera plants comprising an impaired VvMLO7 gene in combinationwith an impaired VvMLO6 or VvMLO11 gene or comprising an impaired VvMLO7gene in combination with impaired VvMLO6 and VvMLO11 genes.

According to a third aspect, the present invention relates to seeds,plant parts or propagation material of the present Erysiphe necatorresistant plants comprising in their genome the present one or twoimpaired Erysiphe necator resistance conferring genes providing Erysiphenecator resistance.

According to a fourth aspect, the present invention relates to isolatednucleotide sequences represented by SEQ ID Nos. 4 or 5 or 6, ornucleotide sequences with more than 70% identity, preferably more than80% identity, more preferably more than 90% identity, and mostpreferably more than 95% identity therewith.

According to a fifth aspect, the present invention relates to isolatedamino acid sequences represented by SEQ ID No. 1 or 2 or 3, or aminoacid sequences with more than 70% identity, preferably more than 80%identity, more preferably more than 90% identity, and most preferablymore than 95% identity therewith.

According to a sixth aspect, the present invention relates to the use ofthe present Erysiphe necator resistance conferring genes, the presentisolated nucleotide sequences or the present isolated amino acidsequences for selecting an Erysiphe necator resistant Vitis viniferaplants using, for example, the present sequences for developingmolecular markers.

The present invention will be further detailed in the following exampleof an especially preferred embodiment of the present invention. In theexample, reference is made to figures wherein:

FIG. 1: shows the area under disease progress curve (AUDPC) ofgrapevines inoculated with Erysiphe necator in control (EVB) andtransgenic lines (TLB1, TLB2, TLB3, TLB4, TLB5, TLB6 and TLB7). The meanscores of AUDPC values calculated on 8-19 biological replicates from twoexperiments are reported. Error bars show standard error of the mean.The asterisks indicates statistically significant differences respect tothe control line EVB, according to Tukey or Games-Howell post-hoc tests(P=0.05). Pictures of representative leaves for each line were collected30 days after inoculation.

FIG. 2: shows Germination of Erysiphe necator conidia in the controlline EVB (A) and in the resistant transgenic line TLB4 (B). Microscopyimages of infected leaves were taken at 3, 10 and 21 days postinoculation (dpi) with powdery mildew. Insert at high magnificationhighlighted the germination of an Erysiphe necator conidia at 3 dpi and10 dpi.

FIG. 3: shows formation of papillae in the control line EVB (A, B) andin the resistant transgenic line TLB4 (C, D). Microscopy images takenwith a bright field (A, C) and fluorescence (B, D) microscope at threedays post inoculation (dpi). The arrows indicate the papillae (P). Thescale bar is the same for the four images.

FIG. 4: shows gene expression of four grapevine MLO genes in the six mlolines (TLB1, TLB2, TLB3, TLB4, TLB5, and TLB6) following inoculationwith Erysiphe necator. Expression of VvMLO6 (A), VvMLO7 (B), VvMLO11 (C)and VvMLO13 (D) was analyzed before (0 dpi; light grey), one (darkgrey), and ten (white) days post inoculation. The mean scores calculatedfrom five to nine plants pooled from the two experiments are reportedfor each line. Error bars show standard error of the mean. For each timepoint, symbols highlight significant differences respect to the controlEVB, according to Tukey or Games-Howell post-hoc test (P=0.05): * for 0dpi, + for 1 dpi and # for 10 dpi.

FIG. 5: shows relative expression of 13 grapevine genes at three timepoints in the control line EVB and in the resistant line TLB4. The colorscale indicates the relative expression values calculated respect to thecontrol EVB at 0 dpi, used as reference for data normalization. Theasterisks highlight statistically significant differences according toFisher post-hoc test. One and two asterisks indicate significance atP=0.05 and P=0.01, respectively. The image was prepared with theMultiexperiment Viewer software with the Log 2 of relative expressiondata

EXAMPLE Materials and Methods Constructs for Grapevine Transformation

Fragments of 300-600 bp for the four MLO target genes VvMLO6, VvMLO7,VvMLO11 and VvMLO13 were amplified with specific primer pairs (Table 1)and cloned into the vector pENTR/SD-TOPO (Invitrogen).

TABLE 1Primers used to amplify MLO genes fragments for the RNAi constructs.Gene^(#) Primer Forward Primer Reverse VvMLO6CACCTGCTTACAGTATTACAAACTCCC TTTCCCTTGCATACCTAAAC VvMLO7CACCGACAATTTTTAACGAGAGAGT ATCTCATGTTGGGTTCGGATT VvMLO11CACCTCACTTATGCTACTGGGGTT ATCAACTTTGGGAACTGATCTGAC VvMLO13CACCGAGCTAATGTTGCTAGGGTT AAATTTTGCATGGCTTTGAG

After sequence validation, the four gene fragments were cloned in theRNAi Gateway vector pK7GWIWG2D(II) (Karimi et al. 2002;http://www.psb.ugent.be/) using the procedure described by Urso et al.(2013). The final constructs were verified by sequencing on both strandsand were cloned into Agrobacterium tumefaciens strain GV3101, asdescribed by Zottini et al. (2008). A. tumefaciens-transformed cellswere tested by PCR (GoTaq Green Master Mix—Promega, Fitchburg, USA) toconfirm the presence of the constructs using specific primers designedto anneal on the 35S promoter (5′-CGCACAATCCCACTATCCTT-3′) and the MLOfragment (Table 1).

Plant Material and Transformation

For grapevine transformation, somatic embryos of V. vinifera cultivarLong-Cluster Brachetto were used. The plant material was in vitrocultivated in the darkness in a growth chamber at 20-24° C. and 70±5%relative humidity (RH). Plant transformation, regeneration and selectionof the transgenic plants were carried out as described by Dalla Costa etal. (2014). A total of five transformations were performed: four aimedto silence the four MLO target genes, one with an empty vector (pK2WG7),as control.

Screening of Regenerants and Propagation of In Vitro Materials

Genomic DNA was extracted from in vitro leaf tissue using IllustraNucleon Phytopure kit (GE Healthcare, Buckinghamshire, UK). Transgeneintegration was evaluated with the same primers used to confirm thepresence of the construct in A. tumefaciens. The in vitro lines thatwere confirmed to have the insertion of the transgene were moved to awoody plant (WP) medium (McCown and Lloyd, 1981), kept in growth chamber(20-24° C., 70±5% RH) and transferred in fresh media once a month.

Greenhouse Acclimation

Plants were acclimated to greenhouse conditions with a progressiveprocess carried out in a growth chamber (25° C., 16 hours day/8 hoursnight, humidity 70±5%). One-month old-plants with a well-developed rootapparatus (at least two main roots, 3 cm long) were transferred in a 250ml plastic cup containing wet autoclaved turf (Terriccio VegetalRadic—Tercomposti Spa, Brescia, Italy) and sealed with parafilm, topreserve humidity. Every seven days, one or two holes were made in theparafilm layer, to progressively reduce the humidity of the environmentand promote the formation of the foliar cuticle. After three weeks, theparafilm sealing was completely removed and, after one week, plants weretransferred into 1 L pots and grown under greenhouse conditions (25° C.,16 hours day/8 hours night, humidity 70±5%).

Erysiphe necator Inoculation and Disease Severity Assessment

The PM inoculum was obtained from infected leaves of an untreatedvineyard in northern Italy (Trentino region) and maintained bysubsequent inoculations on V. vinifera “Pinot Noir” plants undergreenhouse conditions. The plants were dry inoculated with PM by gentlebrushing from infected young leaves carrying freshly sporulation E.necator onto the target leaves (Blaich et al., 1989). Inoculated plantswere incubated in the greenhouse at 25° C. with a relative humidity (RH)of 100% for 6 h to promote the fungal penetration into the leaves, andthen maintained at 25° C. with a relative humidity of 70±10% until thelast symptom's evaluation. Disease severity was visually assessed on allleaves at 14, 22 and 30 days post inoculation (dpi), according to thestandard guidelines of the European and Mediterranean Plant ProtectionOrganisation (EPPO, 1998).

Disease severity was expressed as the proportion (percentage of 0 to100%, with intervals of 5%) of adaxial leaf area covered by PM myceliain relation to the total leaf area, and a mean value was calculated foreach plant. Two inoculation experiments were carried out. For eachexperiment, three to nine biological replicates (plants) per line wereanalyzed in a randomized complete block design. The reduction of diseaseseverity was calculated according to the following formula: [(diseaseseverity in control plants—disease severity in treated plants)/diseaseseverity in control plants]×100. To analyze all the time pointstogether, we used the area under disease progress curve (AUDPC), aquantitative summary of disease intensity over time (Campbell andMadden, 1990; Madden et al., 2007).

To evaluate the disease severity, the number of E. necator conidiaproduced from infected leaves was assessed as described by Angeli et al.(2012) with slight modifications. Three leaves were collected from eachreplicate at 30 dpi and four disks of 0.8 cm diameter for each leaf werecut for a total of 12 disks for replicate. Leaf disks were transferredto 50 mL tubes containing 5 mL distilled water with 0.01% Tween 80(Sigma-Aldrich, Saint Louis, USA). Tubes were vortexed for one min andthe concentration of conidia per ml was determined by counting with ahaemocytometer under a light microscope. The amount of conidia wasfinally converted in conidia per square centimeter (cm²) of grapevineleaf.

Histological Analysis

Two inoculated leaves were collected from three biological replicate ofeach transgenic and control line at 3, 10 and 21 dpi and subjected tohistological analysis. To visualize fungal hyphae, leaves were clearedand stained as described by Vanacker et al. (2000) changed as follow:leaves were cut in small pieces and laid with the adaxial surface up onfilter paper moistened with an ethanol:glacial acetic acid mixture (3:1,v/v) until the chlorophyll had been removed. Leaf pieces weretransferred to water soaked filter paper for 2 h, then transferred on amicroscope slide and a drop of aniline blue (0.1% [w/v] inlactoglycerol) was pipetted onto leaf surface. Hyphae were visualizedusing the bright field illumination of a Leica LMD6500 microscope (LeicaMicrosystems, Wetzlar, Germany).

For the detection of papilla, leaves were cleared in an ethanol:glacialacetic acid mixture (3:1, v/v) until the chlorophyll had been removed,and equilibrated overnight in a solution containing lactic acid,glycerol and water (1:1:1). Papillae were visualized using the LMDfilter (BP filter 380-420 nm excitation, 415 dichroic mirror, and BP445-485 nm emission) of a Leica LMD6500 microscope (Leica Microsystem,Wetzlar, Germany).

Sample Collection, RNA Extraction and Gene Expression Analysis

The first gene expression analysis was carried out on in vitrotransgenic plants, to identify silenced lines, with three biologicalreplicates collected. For the second analysis, carried out on acclimatedtransgenic plants, leaf samples were collected immediately beforeinoculation, 24 hours and 10 days post PM inoculation. These time pointswere chosen because are associated with the up-regulation of MLO genesduring E. necator infection (Feechan et al., 2008; Winterhagen et al.,2008). For each line at each time point, leaf samples were collectedfrom five different plants. Each sample comprised two half leaves takenfrom the same plant; only leaves of the third and fifth nodes from thetop of the shoot were collected. Samples were immediately frozen inliquid nitrogen and stored at 80° C.

Total RNA was extracted with the Spectrum™ Plant Total RNA kit(Sigma-Aldrich), treated with the DNAse I (Sigma-Aldrich) and reversetranscribed using the SuperScript III reverse transcriptase (Invitrogen,Life Technologies, Waltham, USA).

The qPCR analysis was performed with Advanced Universal SYBR GreenSupermix (Bio-Rad, Hercules, USA) in a 15-4, reaction volume withspecific primers (Table 2), using a CFX96 Touch Real-Time PCR detectionsystem (Bio-Rad, Hercules, USA), run by CFX Manager software.

TABLE 2Primers used to amplify MLO genes fragments for the qPCR analysis. NameForward (′5-′3) Reverse (′5-′3) EF1α GAACTGGGTGCTTGATAGGCAACCAAAATATCCGGAGTAAAAGA GAPDH TTCTCGTTGAGGGCTATTCCACCACAGACTTCATCGGTGACA Actin TCCTTGCCTTGCGTCATCTATCACCAATCACTCTCCTGCTACAA VvMLO6 GTGCAGTTATGTGACACTCCC ACACACCATCCGAGTGCVvMLO7 CTTTCTTCGCATGGAGCACG GAGCCCATCTGTGTCACCAA VvMLO11GCACCCCCTTACATGGC TCTGGACCAGGATTTCTATGATG VvMLO13 CTGGTGACACAGATGGGTTCCTACTTGACATGGGTGTGGC VvWRKY19 GGGGAGGCTGTGGTTAGGTT GTTTGGCATTTGGCTTGTCTVvWRKY27 CTTGGATCAGAATCACCCCTAA GCCGTGGTATGTGGTTTTGTA VvWRKY48CAAGATTTCAAGGACCAAGCAG AGTATGCCTTCCTCGGTATGT VvWRKY52CCTCTTGATGATGGGTTTAGTT GTCTTCCACGGTAGGTGATTT VvALS1 CCGTGCATACCGAGCATTTGAGGCCGGTTCTGTATGTTGG VvEDS1 AGGGTTTTATATTGTTATCTCAAGGAAGAAAATATCTTATTACTACATAATG GGC TTTC VvLOX9 GACAAGAAGGACGAGCCTTGCATAAGGGTACTGCCCGAAA VvLOX1 ATCAATGCTCTTGCTCGGGA CCAGAGCTGGTCATAGGCAGVvPAD4 ACGATTGCACTGGTAAGCCA CGACTCCGTCATCGCCTAAA VvPEN1CTTCGCAAGAAGCTCAGGGA TGCTCTTGGATCGCCTTCTG VvPR1 CCCAGAACTCTCCACAGGACGCAGCTACAGTGTCGTTCCA VvPR6 ACGAAAACGGCATCGTAATC TCTTACTGGGGCACCATTTCVvNPF3.2 TCGTCACATCAGCACAGCTT ATCTGCGAGCCAATGGAACA

The software applies comparative quantification with an adaptivebaseline. Samples were run in two technical replicates with thefollowing thermal cycling parameters: 95° C. 3 min, followed by 40cycles of 95° C. 10 sec and 55° C. 30 sec with a final step at 95° C. 10sec. Primers for gene expression analysis of VvMLO6, VvMLO11 and VvMLO13were taken from Winterhagen et al. (2008), while for VvMLO7 we designedour specific primer pair (Table 2). Expression of marker genes modulatedin the interaction between plants and PM were also analyzed. Primers forVvWRKY19, VvWRKY27, VvWRKY48 and VvWRKY52 were taken from Guo et al.(2014), primers for VvEDS1 from Gao et al. (2014) and primers for VvPR1,VvPR6 and VvLOX9 from Dufour et al. (2013). The new primer pairs weredesigned with the NCBI Primer Designing Tool(http://www.ncbi.nlm.nih.gov/tools/primer-blast/) (Table 2). Four serialdilutions of cDNA (1/10-1/100-1/1000-1/10000) were used to calculate theefficiency of the primer pairs and the size of the products wasconfirmed by agarose gel electrophoresis. Presence of a specific finaldissociation curve was determined after every qPCR run with progressiveincrements of temperature from 65° C. to 95° C. (0.5° C. each step, 5sec).

The reference genes were Elongation Factor 1α (GenBank accession numberEC959059), GAPDH (GenBank accession number CB973647) and Actin (GenBankaccession number AY6807019), known to be reference for grapevine (Reidet al., 2006). In this work the stability of these genes was confirmedusing the software GeNorm (medgen.ugent.be/˜jvdesomp/genorm/). All threereference genes had M-values lower than 0.5, when an M-value lower than1.5 was generally considered as stable enough (Ling and Salvaterra,2011; Van Hid et al., 2009; Strube et al., 2008).

The threshold cycles (Ct) were converted to relative expression with thesystem proposed by Hellemans et al. (2007), using as input the averageCt of two technical replicates. Hellemans method takes into account theefficiency value of each primer pair. As reference Ct, we used theaverage Ct of all the samples for the expression of MLO genes, whereasfor the analysis on other genes involved in plant defense or mloresistance, we used the control EVB at T=0. The two different methodswere selected for graphical reasons.

Statistical Analysis Disease Severity

Severity data were analyzed using the Statistica 9 software (StatSoft,Tulsa, USA) and the statistical package SPSS(IBM, Armonk, USA). Thesmallest statistical unit was the whole plant. We averaged the severityvalues of all the leaves of the plant and used the resulting averageseverity for further analysis. Data with a normal distribution(Kolmogorov-Smirnov and Shapiro-Wilk tests P>0.05) were validated forvariances homogeneity (Levene's test, P>0.05) and subsequently used forone-way ANOVA with Tukey's post-hoc test, to detect significantdifferences (P<0.05) at each time point. Data were transformed with thefollowing equation y=arcsin(x), in order to meet the pre-requisites ofthe ANOVA. In case of non-homogeneous variances, the Games-Howell'spost-hoc test was used.

Data from the two experiments were pooled, and the analysis carried outindependently for the three time points (14, 22 and 30 dpi). AUDPC datawere analyzed as described above for severity data. Data of the conidiacounts were analyzed with Kruskall-Wallis test (P<0.05).

qPCR Data Analysis

For the gene expression analysis, values of relative expression weretransformed in logarithmic scale according to the equation Y=ln(x)(Pessina et al., 2014) to obtain normal distribution and homogeneity ofvariances of the residues, assessed with the tests of Shapiro-Wilk(P<0.05) and Levene (P<0.05), respectively. Pairwise comparison ofhomoscedastic data was carried out with Tukey's test (P<0.05), whereasnon-homoscedastic data were analyzed with Games-Howell test (P<0.05)using the statistical package SPSS(IBM).

The relative expression of MLO genes from two experiments were analyzedindependently and subsequently pooled. To assess differences inexpression, one-way ANOVA with Tukey post-hoc test (P<0.05) was used todetect significant differences at each time point. In addition, atwo-way ANOVA with Tukey post-hoc test (P<0.05) was used on all thedata, to consider at the same time the effects of the transgenic lineand of the time point. We drew conclusions from this test only whenthere were no significant interactions (P>0.05) between the factors timepoint and transgenic line. For the gene expression characterization ofTLB4, we used Fisher as post-hoc test.

Correlation

We used the two-tailed Pearson's correlation test to investigate twopossible correlations: between disease severity and amount of conidia at30 dpi and between disease severity at 14 dpi and relative expression ofMLO genes at 10 dpi. In both cases, all data, severity and relativeexpression, have been transformed with the following equationy=arcsin(x), to achieve normal distribution.

Results Transformation, Selection and Acclimation of MLO TransgenicLines

A total of five gene transfers were carried out. Four were aimed toknock-down (KD) specific MLO genes (i=KD-VvMLO6, ii=KD-VvMLO7,iii=KD-VvMLO11, iv=KD-VvMLO13), the fifth to insert an empty vector.Thirty-four regenerated lines were obtained, with 26 of them confirmedto contain the insert (Table S3). The result of the PCR analysis of sixlines is shown in Fig. S1. Twenty-six transgenic lines were propagatedin vitro and tested for the silencing of MLO genes with qPCR. This wasevident for three lines out of eight from gene transfer (iii)(KD-VvMLO11), and three out of nine from gene transfer (iv)(KD-VvMLO13). Gene transfers (i) (KD-VvMLO6) and (ii) (KD-VvMLO7)resulted in a small number of regenerated lines that showed no reductionof expression (Table S3). Regenerated lines were also tested foroff-target silencing, showing that the RNAi fragments targeted otherclade V MLO genes. Six lines with various combinations of silenced geneswere selected and indicated with acronims TLB1 (Transgenic Line ofBrachetto) to TLB6 (Table S3). Lines from TLB1 to 3 came from genetransfer (iii) (KD-VvMLO11), lines from TLB4 to TLB6 from gene transfer(iv) (KD-VvMLO13) (Table S3). The control was the EVB line (Empty VectorBrachetto). In addition, TLB7, a regenerated line with no reduction ofexpression, was also considered. All lines, including the control, willbe referred in the text as “transgenic lines”. Lines from TLB1 to 7 arefurther indicated as “RNAi lines” and from TLB1 to 6 “mlo lines”.

The survival rate of plants to the acclimation process was around 85%.Under greenhouse conditions, the transgenic plants showed normal growthand no pleiotropic phenotypes.

Powdery Mildew Resistance of Transgenic Lines

PM inoculation was carried out on the seven RNAi lines (TLB1, TLB2,TLB3, TLB4, TLB5, TLB6, TLB7), and the transgenic control line EVB intwo independent experiments. Three mlo lines, TLB4, TLB5 and TLB6,showed a significant reduction of E. necator infection (FIG. 1) whichwas greater than 60% at 30 dpi (Table 3).

TABLE 3 Disease reduction of seven RNAi lines. Average Number of Diseasereduction %* Disease reduction plants 14 dpi 22 dpi 30 dpi (%) TLB1 822.8 32.3 34.3 29.8 TLB2 15 49.2 37.2 23.8 36.8 TLB3 15 17.9 14.8 2.011.6 TLB4 19 60.8 71.7 72.8 68.4 TLB5 14 76.7 79.1 74.0 76.6 TLB6 1171.8 63.1 60.3 65.1 TLB7 13 −8.0^(#) −21.5^(#) −21.2^(#) −16.9^(#) *LineEVB was used as control (12 replicates) and disease reduction wascalculated as (Disease severity of EVB - disease severity of thetransgenic line)/disease severity of EVB × 100. ^(#)The negative valuesof TLB7 mean that the line showed higher level of infection compared toEVB

The disease reduction of TLB6 decreased with the progression of theinfection (Table 3), possibly because of the secondary infections comingfrom nearby infected plants. TLB2, TLB3, and TLB7 showed a level ofsusceptibility to PM comparable to the EVB control (FIG. 1 and Table 2).The leaves in FIG. 1 showed the differences between resistant andsusceptible lines. All the mlo lines showed reduction of conidia on theleaves surface at 30 dpi, and the decrease was statistically significantonly for TLB4, TLB5 and TLB6. TLB4 showed a reduction of 93% of conidia,TLB5 of 95% and TLB6 of 72% compared to the EVB plants. The conidiacounts and the disease severity were positively correlated (P=0.01),with a Pearson correlation coefficient of 0.578. This means that thereduction of symptoms on the leaves reflected the lower number ofconidia on the resistant lines.

Line TLB4 was further characterized by histological analysis,demonstrating a reduced progression of PM infection compared to EVBplants at 3 dpi (FIG. 2). In EVB, the first conidiophores appeared at 10dpi, and at 21 dpi they were spread all over the leaf surface (FIG. 2A).On the other hand, conidiophores were visible only at 21 dpi and in alimited number on TLB4 leaves (FIG. 2B). The formation of papilla wasobserved in TLB4 and EVB at 3 dpi (FIG. 3). The papilla of EVB haddefined edges and it was present only in correspondence of the infectionsite of E. necator (FIG. 3B). Conversely, the papilla detected in TLB4was more diffuse, bigger and formed in more than one site of infectionof the fungus compared to EVB (FIG. 3D).

Expression of MLO Genes in the MLO Transgenic Lines and Correlation withSeverity

Six mlo lines (TLB1, TLB2, TLB3, TLB4, TLB5, TLB6) and the control EVBwere examined by gene expression analysis. Gene expression analysis ofthe four clade V MLO genes in the transgenic lines confirmed theoff-target silencing seen in vitro and showed some variability amongtime points (FIG. 4). Lines TLB1, TLB2 and TLB3, all transformed withthe constructe aimed to silence VvMLO11, indeed had the target geneVvMLO11 silenced. TLB1 showed also the silencing of VvMLO13 and TLB3 ofVvMLO6 (Table 4).

TABLE 4 Relative expression^(#) of four MLO genes VvMLO6 VvMLO7 VvMLO11VvMLO13 TLB1 67% 72% 25%** 49%** TLB2 79% 94% 40%** 156%   TLB3 71%* 93%27%** 69%  TLB4 38%** 49%** 34%** 33%** TLB5 35%** 55%** 50%** 88%  TLB642%** 53%** 55%** 45%** ^(#)Each relative expression (RE %) value is theaverage of the values of three time points (0 dpi, 1 dpi, 10 dpi) in twoexperiments. RE % was calculated as follow: RE % = (RE of control EVB/REof mlo line) * 100. *statistically significant difference at P = 0.05,accordint to Tukey post-hoc test. **statistically significant differenceat P = 0.01, accordint to Tukey post-hoc test.

Lines TLB4, TLB5 and TLB6, coming from the transformation aimed tosilence VvMLO13, showed more off-target silencing. In TLB4 and TLB6, allfour clade V MLO genes were silenced, whereas In TLB5 VvMLO6, VvMLO7 andVvMLO11 were silenced (Table 4).

A statistically significant (P=0.05) positive Pearson's correlation wasfound between the relative expression of VvMLO7 and the severity of PMsymptoms, but not for the other three MLO genes. The Pearson correlationcoefficiency for VvMLO7 was 0.272, meaning that the correlation,although significant, was weak.

Gene Expression Analysis of the Mlo Line TLB4

The expression profile of 13 genes known to be modulated following PMinfections was carried out on the resistant line TLB4 at three timepoints (FIG. 5). Line TLB4 was selected because it has all four MLOclade V genes silenced. In EVB, we observed a general up-regulation ofgenes, especially at 10 dpi. Instead, in the transgenic line TLB4, fewergenes were up-regulated and the intensity of up-regulation, in terms offold-change, was limited. Moreover, three genes were down-regulated inTLB4 after inoculation, namely VvPR6 (PATHOGENESIS RELATED) at 1 dpi andVvNPF3.2 (NITRATE TRANSPORTER/PEPTIDE TRANSPORTER FAMILY) and VvALS1(ACETOLACTATE SYNTHASE) at 10 dpi. It is noteworthy that, before theinoculation, there were no differences in expression between TLB4 andthe control EVB.

DISCUSSION

Loss-of-function mutations of MLO genes reduce susceptibility to PM inbarley (Büschges et al., 1997), Arabidopsis (Consonni et al., 2006), pea(Pavan et al., 2011), tomato (Bai et al., 2008), wheat (Wang et al.,2014), and pepper (Zheng et al., 2013). Because in dicots all Clade VMLO S-genes are implicated in PM susceptibility (Consonni et al., 2006;Bai et al., 2008; Feechan et al., 2008; Winterhagen et al., 2008), theaim of this work was to identify which of the clade V MLO genes ofgrapevine has a role in PM susceptibility, and can thus be inactivatedto develop resistant genotypes. Out of 26 transgenic lines, six fromgene transfers (iii) (KD-VvMLO11) and (iv) (KD-VvMLO13) supportedsignificant gene knock-down. In the regenerated lines obtained from genetransfers (i) (KD-VvMLO6) and (ii) (KD-VvMLO7), reduction of expressionwas not evident. It cannot be excluded that this was due to the shortRNAi fragments present in the constructs (Preuss and Pikaard, 2003). Thedetection of off-target silencing in five of the six mentioned lines wasexpected, as clade V MLO genes have high levels of sequence identity(36-60%, 46% on average; Feechan et al., 2008; Winterhagen et al.,2008). To find a balance between specificity (short RNAi fragments) andeffectiveness (long RNAi fragments) is particularly difficult in genefamilies with high sequence similarity (Zhao et al., 2005). Since theaim was to study the effect of the knock-down of four MLO genes quitesimilar to each other, we opted for long RNAi fragments, so thatoff-target silencing was not only expected, but also desired. Knock-outand knock-down of MLO genes may induce pleiotropic phenotypes, likenecrotic spot on leaves and reduced grain yield in barley (Jorgensen,1992), slow growth in Arabidopsis (Consonni et al., 2006) and reducedplant size in pepper (Zheng et al., 2013). In grapevine, no pleiotropicphenotypes were observed under the experimental conditions adopted.Lines TLB4, 5 and 6, which showed clear resistance to PM, allowed tostudy the link between resistance and the expression of specific MLOgenes. VvMLO11 expression was significantly reduced in susceptible andresistant mlo lines: it is concluded that its knock-down was notdirectly linked to grapevine susceptibility to PM. VvMLO6 wassignificantly silenced in the resistant lines TLB4, 5 and 6 and in thesusceptible line TLB3. Like for VvMLO11, the knock-down of VvMLO6 inboth susceptible and resistant lines indicates that this should not be aS-gene. Similarly to VvMLO6, VvMLO13 was knocked-down in the resistantlines TLB4 and 6, but also in the susceptible line TLB1. VvMLO7 wasknocked-down only in the three resistant lines TLB4, 5 and 6; theconclusion is that VvMLO7 represents the main candidate for causing PMsusceptibility in V. vinifera. The significant positive correlationbetween the relative expression of VvMLO7 and the disease severity inthe MLO transgenic lines, stimulates the conclusion that either sitedirected mutagenesis or searching for natural non-functional alleles maybe used in breeding programs to obtain PM resistant genotypes. It was,however, noted that VvMLO7 was always knocked-down together with othertwo or three MLO genes. Also in Arabidopsis the contemporary knock-outof three MLO genes is necessary to obtain complete resistance: knock-outof AtMLO2 results in a moderate level of resistance, whereas knock-outof AtMLO6 and AtMLO12, alone or combined, does not decrease theintensity of the infection. When AtMLO2 is knocked-out together withAtMLO6 or AtMLO12, the level of resistance rises, to become completewhen the three genes are knocked-out together (Consonni et al., 2006).In grapevine, VvMLO7 seemed to act like AtMLO2 of Arabidopsis. Twocandidates for an additive and synergistic role in PM susceptibility ingrapevine are VvMLO6 and VvMLO11, since their expression wassignificantly reduced in all three resistant lines. In Arabidopsis, theknock-out of three MLO genes induces complete resistance (Consonni etal., 2006), a situation not observed in grapevine, in agreement with theincomplete silencing of MLO genes obtained by the RNAi approach. Acomplementation test, carried out in Arabidopsis mlo triple mutant,showed that VvMLO11 and VvMLO13 induce susceptibility to PM, whereasVvMLO7 has only a partial effect and VvMLO6 has no effect at all(Feechan et al., 2013b). However, single and double VvMLO11 and VvMLO13knock-down mutants of V. vinifera obtained by RNAi, did not showsignificant reduction of PM penetration (Qiu et al., 2015). Accordingly,our data indicated VvMLO7 as the main S-gene of grapevine, with aputative additive effect provided by VvMLO11 and VvMLO6. The role ofVvMLO6 would be particularly surprising, as it was not up-regulatedduring PM infection (Feechan et al., 2008; Winterhagen et al., 2008).Conversely, VvMLO13, which knock-down was expected to provide asignificant effect on PM susceptibility, turned out to be ineffective.However, it should be considered that Feechan et al. (2013b) operated ina heterologous system (Arabidopsis) not reproducing with fidelity the PMinfection of grapevine plants.

The precise mechanism through which the reduction of MLO genesexpression ends up in resistance to PM pathogens is not completelyclear. Resistance seems linked to secretory vesicles traffic (Miklis etal., 2007; Feechan et al., 2011) and to the formation of cell wallappositions called papillae (Consonni et al., 2006). These structuresconsists of a callose matrix enriched in proteins and autofluorogenicphenolics compounds (Vanacker et al. 2000), and their formation dependson endomembrane transport (Hückelhoven, 2014). The results shown in thispaper indicate that all transgenic lines accumulate autofluorigenicmaterials overimposed to the papilla structure, although shape anddimensions of papillae were different in resistant and susceptiblelines. It is known that the defense response based on papillae differsbetween resistant and susceptible genotypes in timing of formation,composition and size (Chowdhury et al., 2014; Hückelhoven, 2014; Lyngkj

et al. 2000). Rapid formation of papillae in mlo resistant barley(Lyngkj

et al. 2000) and increased size (Stolzenburg et al., 1984) correlatewith mlo resistance. In grapevine, papilla formation is restricted tothe site of infection in control plants, whereas it is diffused in theresistant line TLB4. Chowdhury et al. (2014) showed that the differencebetween effective and non-effective papillae is due to the higherconcentration of callose, cellulose and arabinoxylan of the effectiveones. This suggests that, in the case of grapevine, different types offluorescence could reflect differences in the composition of thepapillae. The MLO protein has been proposed to be a negative regulatorof vesicle-associated and actin-dependent defense pathways at the siteof attempted PM penetration (Panstruga, 2005). Furthermore, Miklis etal. (2007) proposed that, once MLO proteins are under the control of thefungus, actin filaments serve the purpose of supplying nutrients for thegrowing hyphae through vesicular transport. It is like if the pathogenis able to control the transport of material to the cell-wall, with thepurpose of changing the composition of the papillae and turning themfrom effective to non-effective. The formation of papillae is not theonly process instigated by the activity of MLO genes. To understand theeffect of MLO knock-down on other genes involved in plant-pathogeninteraction, the expression of 13 genes known to be differentiallyexpressed after PM inoculation was analyzed. In the resistant line TLB4,the knock-down of MLO genes did not affect the level of expression ofthe 13 genes in absence of PM infection. Under E. necator infection (Guoet al., 2014), transcription factors VvWRKY19, VvWRKY48 and VvWRKY52 areup-regulated: the same genes appeared up-regulated in EVB in ourexperiments, but they were so at a much lower level in TLB4. VvNPF3.2, anitrite/nitrate transporter up-regulated in grapevine infected with E.necator (Pike et al., 2014), was down-regulated in TLB4 at 10 dpi,indicating that in this line only a severe infection elicits VvNPF3.2up-regulation. VvEDS1 (enhanced disease susceptibility) and VvPAD4(phytoalexin deficient) are grapevine defense genes involved in thesalicylic acid (SA) pathway (Gao F. et al., 2014). SA activatespathogenesis related genes and induces disease resistance (Ward et al.,1991). Both genes were up-regulated in the control line EVB at 10 dpi(VvPAD4 also at 1 dpi). This may indicate that only a heavy E. necatorinfection triggers the plant defense depending on SA. VvEDS1 was notup-regulated in TLB4, whereas VvPAD4 was up-regulated only at 10 dpi,like if the level of PM infection was insufficient to activate thereaction of the plant. Up-regulation in the control and no up-regulationin TLB4 was also observed for both VvPR1 and VvPR6, pathogenesis-relatedgenes involved in plant defense and known to be up-regulated in PMinfected grapevine leaves pre-treated with an SA analogue (Dufour etal., 2013). VvLOX1 encodes a lipoxygenase and is the homologous toArabidopsis AtLOX2, that is up-regulated in plants infected with PMspores (Lorek, 2012). Surprisingly, this gene was up-regulated in TLB4at 10 dpi, but not in EVB. A second lipoxygenase, VvLOX9, did not showin the grapevine lines considered any change in expression, despitebeing known to be involved in plant defense (Dufour et al., 2013).VvPEN1 (penetration) encodes for a SNARE protein homologous toArabidopsis AtPEN1 and barley ROR2, which have important roles in PMpenetration resistance (Collins et al., 2003). VvPEN1 when expressed ina heterologous system (Arabidopsis), is known to co-localize withVvMLO11 at sites of attempted PM penetration (Feechan et al., 20013b).However, infection with E. necator did not cause any change of itsexpression. VvALS1 is the homologous of a tomato acetolactate synthase,a key enzyme in the biosynthesis of the amino acids valine, leucine andisoluecine, and involved in mlo-mediated resistance (Gao D. et al.,2014). Silencing of SlALS1 in mlo tomato compromises its resistance,suggesting that amino acid homeostasis is an important process connectedto mlo resistance (Gao D. et al., 2014). The complete lack oftranscriptional change indicated that the function of this gene ingrapevine does not depend on the transcript level. The knock-out of MLOgenes increased susceptibility to other pathogens in barley (Jarosch etal., 1999; Kumar et al., 2001) and Arabidopsis (Consonni et al., 2006).The infection with P. viticola, an obligate biotroph fungus like E.necator, revealed that the knock-down of MLO genes did not change thesusceptibility of grapevine to downy mildew, supporting the conclusionthat MLOs S-genes are specific for E. necator and are not involved inthe plant interaction with P. viticola.

CONCLUSIONS

The knock-down of MLO genes substantially reduces PM susceptibility ofVitis vinifera. The reduction of expression of VvMLO7 was the mainfactor involved in resistance, but the additive effects of VvMLO6 andVvMLO11 knock-down further contribute in reducing PM severity. Absoluteresistance was not observed, as expected based on the incompletesilencing of MLO genes via RNAi. In mlo lines, no pleiotropic phenotypeswere detected under greenhouse conditions. This work provides a crucialinformation that can be used in breeding grapevine varieties resistantto E. necator. The tagging via genome editing of the MLO genesidentified in this paper, particularly of VvMLO7, should results inknock-out mutants highly resistant to PM. Alternatively, the search inV. vinifera and in wild species of non-functional MLO alleles,particularly of VvMLO7, should contribute to the creation of durableresistance.

ABBREVIATIONS

AUDPC: area under disease progress curvedpi: days post inoculationEVB: empty vector BrachettoPM: powdery mildewRE: relative expressionSA: salicylic acid

TLB1-7=Transgenic Line Brachetto 1-7 REFERENCES

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1-21. (canceled)
 22. An isolated Vitis vinifera having resistance topowdery mildew and comprising in its genome a modified gene comprisingone or more non-natural mutations, insertions, substitutions, ordeletions, wherein the modified gene results in: a decrease of function,loss of function, reduced expression, or absence of a protein having theamino acid sequence of SEQ ID NO: 1 compared to a Vitis vinifera lackingsaid modified gene, or a decrease in mRNA having the nucleotide sequenceof SEQ ID NO: 4 compared to a Vitis vinifera lacking said modified gene.23. The isolated Vitis vinifera of claim 22, wherein the resistance topowdery mildew is resistance to powdery mildew caused by Erysiphenecator.
 24. The isolated Vitis vinifera of claim 22, wherein themodified gene results in at least a 10% reduction in expression of theprotein having the amino acid sequence of SEQ ID NO: 1 compared to aVitis vinifera lacking said modified gene or at least a 10% reduction inamount of mRNA having the nucleotide sequence of SEQ ID NO: 4 comparedto a Vitis vinifera lacking said modified gene.
 25. The isolated Vitisvinifera of claim 22, further comprising in its genome a second modifiedgene comprising one or more non-natural mutations, insertions,substitutions, or deletions, wherein the modified second gene resultsin: a decrease of function, loss of function, reduced expression, orabsence of a protein having the amino acid sequence of SEQ ID NO: 2compared to a Vitis vinifera lacking said second modified gene, or adecrease in mRNA having the nucleotide sequence of SEQ ID NO: 5 comparedto a Vitis vinifera lacking said second modified gene.
 26. The isolatedVitis vinifera of claim 22, further comprising in its genome a secondmodified gene comprising one or more non-natural mutations, insertions,substitutions, or deletions, wherein the modified second gene resultsin: a decrease of function, loss of function, reduced expression, orabsence of a protein having the amino acid sequence of SEQ ID NO: 3compared to a Vitis vinifera lacking said second modified gene, or adecrease in mRNA having the nucleotide sequence of SEQ ID NO: 6 comparedto a Vitis vinifera lacking said second modified gene.
 27. The isolatedVitis vinifera of claim 22, further comprising in its genome second andthird modified genes comprising one or more non-natural mutations,insertions, substitutions, or deletions, wherein the modified secondgene results in: a decrease of function, loss of function, reducedexpression, or absence of a protein having the amino acid sequence ofSEQ ID NO: 2 compared to a Vitis vinifera lacking said second modifiedgene, or a decrease in mRNA having the nucleotide sequence of SEQ ID NO:5 compared to a Vitis vinifera lacking said modified gene; and whereinthe modified third gene results in: a decrease of function, loss offunction, reduced expression, or absence of a protein having the aminoacid sequence of SEQ ID NO: 3 compared to a Vitis vinifera lacking saidthird modified gene, or a decrease in mRNA having the nucleotidesequence of SEQ ID NO: 6 compared to a Vitis vinifera lacking said thirdmodified gene.
 28. A seed, fruit, plant part, or propagation material ofthe isolated Vitis vinifera of claim 22, wherein the seed, fruit, plantpart, or propagation material comprises the modified gene comprising oneor more non-natural mutations, insertions, substitutions, or deletionsresulting in: a decrease of function, loss of function, reducedexpression, or absence of a protein having the amino acid sequence ofSEQ ID NO: 1 compared to a Vitis vinifera lacking said modified gene, ora decrease in mRNA having the nucleotide sequence of SEQ ID NO: 4compared to a Vitis vinifera lacking said modified gene.
 29. A seed,fruit, plant part, or propagation material of the isolated Vitisvinifera of claim 25, wherein the seed, fruit, plant part, orpropagation material comprises the modified gene comprising one or morenon-natural mutations, insertions, substitutions, or deletions resultingin: a decrease of function, loss of function, reduced expression, orabsence of a protein having the amino acid sequence of SEQ ID NO: 1compared to a Vitis vinifera lacking said modified gene, or a decreasein mRNA having the nucleotide sequence of SEQ ID NO: 4 compared to aVitis vinifera lacking said modified gene, and wherein the seed, fruit,plant part, or propagation material comprises the second modified genecomprising one or more non-natural mutations, insertions, substitutions,or deletions resulting in: a decrease of function, loss of function,reduced expression, or absence of a protein having the amino acidsequence of SEQ ID NO: 2 compared to a Vitis vinifera lacking saidsecond modified gene, or a decrease in mRNA having the nucleotidesequence of SEQ ID NO: 5 compared to a Vitis vinifera lacking saidsecond modified gene.
 30. A seed, fruit, plant part, or propagationmaterial of the isolated Vitis vinifera of claim 26, wherein the seed,fruit, plant part, or propagation material comprises the modified genecomprising one or more non-natural mutations, insertions, substitutions,or deletions resulting in: a decrease of function, loss of function,reduced expression, or absence of a protein having the amino acidsequence of SEQ ID NO: 1, or a decrease in mRNA having the nucleotidesequence of SEQ ID NO: 4 compared to a Vitis vinifera lacking saidmodified gene, and wherein the seed, fruit, plant part, or propagationmaterial comprises the second modified gene comprising one or morenon-natural mutations, insertions, substitutions, or deletions resultingin: a decrease of function, loss of function, reduced expression, orabsence of a protein having the amino acid sequence of SEQ ID NO: 3compared to a Vitis vinifera lacking said second modified gene, or adecrease in mRNA having the nucleotide sequence of SEQ ID NO: 6 comparedto a Vitis vinifera lacking said second modified gene.
 31. A seed,fruit, plant part, or propagation material of the isolated Vitisvinifera of claim 27, wherein the seed, fruit, plant part, orpropagation material comprises the three modified genes comprising oneor more non-natural mutations, insertions, substitutions, or deletionsresulting in: a decrease of function, loss of function, reducedexpression, or absence of a protein having the amino acid sequence ofSEQ ID NO: 1 compared to a Vitis vinifera lacking said modified gene, ora decrease in mRNA having the nucleotide sequence of SEQ ID NO: 4compared to a Vitis vinifera lacking said modified gene, and wherein theseed, fruit, plant part, or propagation material comprises the secondmodified gene comprising one or more non-natural mutations, insertions,substitutions, or deletions resulting in: a decrease of function, lossof function, reduced expression, or absence of a protein having theamino acid sequence of SEQ ID NO: 2 compared to a Vitis vinifera lackingsaid second modified gene, or a decrease in mRNA having the nucleotidesequence of SEQ ID NO: 5 compared to a Vitis vinifera lacking saidsecond modified gene, and wherein the seed, fruit, plant part, orpropagation material comprises the third modified gene comprising one ormore non-natural mutations, insertions, substitutions, or deletionsresulting in: a decrease of function, loss of function, reducedexpression, or absence of a protein having the amino acid sequence ofSEQ ID NO: 3 compared to a Vitis vinifera lacking said third modifiedgene, or a decrease in mRNA having the nucleotide sequence of SEQ ID NO:6 compared to a Vitis vinifera lacking said third modified gene.
 32. Amethod for obtaining a Vitis vinifera having resistance to powderymildew comprising introducing a modification comprising one or snorenon-natural mutations, insertions, substitutions, or deletions to a geneencoding a protein having the amino acid sequence of SEQ ID NO: 1 in aVitis vinifera, wherein the modification results in: a decrease offunction, loss of function, reduced expression, or absence of a proteinhaving the amino acid sequence of SEQ ID NO: 1 compared to a Vitisvinifera lacking the modified gene, or a decrease in mRNA having thenucleotide sequence of SEQ ID NO: 4 compared to a Vitis vinifera lackingsaid modified gene.
 33. The method of claim 32, wherein the resistanceto powdery mildew is resistance to powdery mildew caused by Erysiphenecator.
 34. The method of claim 32, wherein the modified gene resultsin at least a 10% reduction in expression of the protein having theamino acid sequence of SEQ ID NO: 1 compared to a Vitis vinifera lackingsaid modified gene or at least a 10% reduction in amount of mRNA havingthe nucleotide sequence of SEQ ID NO: 4 compared to a Vitis viniferalacking said modified gene.
 35. The method of claim 32, wherein themethod further comprises introducing a second modification comprisingone or more non-natural mutations, insertions, substitutions, ordeletions to a second gene encoding a protein having the amino acidsequence of SEQ ID NO: 2 in a Vitis vinifera, wherein the modificationresults in: a decrease of function, loss of function, reducedexpression, or absence of a protein having the amino acid sequence ofSEQ ID NO: 2 compared to a Vitis vinifera lacking the second modifiedgene, or a decrease in mRNA having the nucleotide sequence of SEQ ID NO:5 compared to a Vitis vinifera lacking the second modified gene.
 36. Themethod of claim 32, wherein the method further comprises introducing asecond modification comprising one or more non-natural mutations,insertions, substitutions, or deletions to a second gene encoding aprotein having the amino acid sequence of SEQ ID NO: 3 in a Vitisvinifera, wherein the modification results in: a decrease of function,loss of function, reduced expression, or absence of a protein having theamino acid sequence of SEQ ID NO: 3 compared to a Vitis vinifera lackingthe second modified gene, or a decrease in mRNA having the nucleotidesequence of SEQ ID NO: 6 compared to a Vitis vinifera lacking the secondmodified gene.
 37. The method of claim 32, wherein the method furthercomprises: introducing a second modification comprising one or morenon-natural mutations, insertions, substitutions, or deletions to asecond gene encoding a protein having the amino acid sequence of SEQ IDNO: 2 in a Vitis vinifera, wherein the modification results in: adecrease of function, loss of function, reduced expression, or absenceof a protein having the amino acid sequence of SEQ ID NO: 2 compared toa Vitis vinifera lacking the second modified gene, or a decrease in mRNAhaving the nucleotide sequence of SEQ ID NO: 5 compared to a Vitisvinifera lacking the second modified gene; and introducing a thirdmodification comprising one or more non-natural mutations, insertions,substitutions, or deletions to a third gene encoding a protein havingthe amino acid sequence of SEQ ID NO: 3 in a Vitis vinifera, wherein themodification results in: a decrease of function, loss of function,reduced expression, or absence of a protein having the amino acidsequence of SEQ ID NO: 3 compared to a Vitis vinifera lacking the secondmodified gene, or a decrease in mRNA having the nucleotide sequence ofSEQ ID NO: 6 compared to a Vitis vinifera lacking the second modifiedgene.